The clinical amelioration in sickle cell disease seen with persistent fetal hemoglobin expression should be explored for this disease. Gene transfer of a transcription factor capable of reversing the gamma to beta globin switch in adult erythroid cells represents one approach to the treatment of this devastating disorder. n advantage of this strategy over the transfer of cis-sequences encoding the globin genes is the concomitant reduction of beta/S expression that would be anticipated. The long-term goal of this project is to identify a factor(s) which may be utilized in this approach to gene therapy. Over the current funding period we have identified three factors which may fulfill this criteria. Firstly, we have identified a fetal and erythroid component of the stage selector protein complex. This complex has been implicated in the recruitment of the locus control region to the gamma promoter during fetal erythropoiesis. This complex has studies proposed in our first specific aim seek to define the role of this factor, c106, in the formation of the SSP complex and determine its role in fetal erythroid cells. We will evaluate the ability of the factor to activate fetal hemoglobin expression in a novel model of primitive and definitive erythropoiesis, mice transgenic for the human beta globin locus and human erythroid progenitors. Ultimately, if these studies demonstrate that c106 is capable of reactivating fetal hemoglobin expression, its ability to modulate gamma-globin gene expression in a NOD/SCID model of sickle erythropoiesis and non-human primates will be determined in collaboration with Projects 4 and 5. In addition, we have also developed a novel screening strategy to identify further fetal globin regulatory genes utilizing novel chemical probes. With this strategy, as outlined in the work proposed in specific aim 2, we have identified two factors, the HLH protein Id2 and a novel CCAAT box binding protein homologue Hap5h. Interestingly, enforced expression of Id2 in fetal erythroid cells results in a 9 fold induction of gamma globin gene expression. We propose to characterize these factors further, to determine their role in the gamma to beta switch, as well as their potential therapeutic role. Finally, in specific aim 3 we describe the use of further novel chemical probes with diverse structures that allow us to identify further fetal regulatory factors. In summary, these studies will enhance our understanding of the molecular regulation of gamma globin gene expression and may provide new therapeutic strategies for the treatment of sickle cell disease.